Hydrolases are enzymes which have recently achieved great importance. On the one hand, they play an important role in the metabolism of plants and animals and also of humans. If the concentration of a hydrolase in one of these biological systems deviates from the concentration normally present therein, then this can be the cause of a disease. Therefore, it is .2 task of clinical diagnosis, in the case of the presence of a disease, to ascertain, by determination of the concentration of a hydrolase in body fluids, possible deviations from the normal value. This preferably takes place via the determination of the hydrolase activity by means of an indicator reaction. For this purpose, the sample to be investigated is mixed with a substrate for the hydrolase in question. The amount of product formed from the substrate is a measure for the hydrolase concentration present.
On the other hand, hydrolases are used ever more frequently as enzymes for labeling immunologically active compounds. .beta.-Galactosidase is hereby especially widely used for the labelling of, for example, antibodies in immunological tests (see Annals of Clinical Biochemistry, 16, 221/1979). Tests of this kind serve to determine the content of an immunologically active analyte in a sample. They are so constructed that the concentration of the analyte is determined via an appropriate immune component which carries covalently bound .beta.-galactosidase as label. The test is so constructed that the concentration of the immune partner is directly dependent upon the concentration of the analyte to be determined. The concentration of the labelled immune component is also made visible via an indicator reaction, in which the immune component labelled with .beta.-galactosidase is reacted with a substrate for .beta.-galactosidase.
The amount of product formed is proportional to the concentration of the immune component. By comparison with the values of a calibration curve, which is produced with the help of samples with known analyte concentration, an unknown analyte concentration in a sample can be determined.
Hydrolases are also used as enzymes for labelling nucleic acids in processes for the detection of nucleic acids. Such a process which uses .beta.-galactosidase as enzyme label is described in Federal Republic of Germany Patent Specification No. 29 15 082. Here, too, the amount of label is determined in an indicator reaction.
Furthermore, hydrolases are used as reagents in research. Here, too, it is important to be able to determine the concentration of the enzymes exactly and quickly. In general, for this purpose, there are used methods which are similar to those used in clinical diagnosis.
The substrate used is generally a chemical compound which is soluble in the sample liquid. In the case of the reaction with the hydrolase, a product is formed which differs in one of its characteristics, for example a characteristic light absorption, a light emission (fluorescence) or the like, from the substrate and can thereby be determined.
In the case of test processes on immunologically active substances, a differentiation is made between two technical embodiments. In the case of a frequently used embodiment, the detection reaction takes place in a cuvette such as is generally used for photometric investigations. The evaluation of the indicator reaction is then carried out by measurement of the absorption, emission or radio-activity in an appropriate apparatus.
In the case of another embodiment, the reactions take place in one or more fleece or films which are parts of a test carrier, the necessary reagents being applied to these fleece or films. The amount of product which is liberated by the hydrolase activity from the hydrolase substrate during the indicator reaction can then be determined directly in such a fleece or film. An advantage of this embodiment is that it is possible to work with only a single solution, the liquid sample, which considerably simplifies the carrying out of the process. The amount of product formed can be detected especially simply by measurement of the light absorption at a particular wavelength.
Substrates suitable for this purpose include the phenyl-sulphophthaleinyl-.beta.-D-galactosides and derivatives thereof, which are described in European Patent Specification No. 01 46 866, from which phenylsulphophthalein derivatives are liberated by reaction with .beta.-D-galactosidase. Whereas the substrate has a yellow color, the product is red colored. Thus, during the indicator reaction, a gradual color change from yellow via orange to red takes place. The color change can only be assessed very inaccurately with the naked eye so that an appropriate photometer must be used for the measurement.
The resorufin-.beta.-D-glycosides described in European Patent Specification No. 0 156 347 are also substrates in which case the reaction with a glycosidase causes a color change from yellow to red to take place. It has been ascertained that in the transitional region between yellow and red, especially in the case of low .beta.-glycosidase activities, the visual evaluation leads to subjective results, for which reason apparatus are here also used for the evaluation. However, these apparatus are relatively complicated and, therefore, expensive. The resorufin glycosides are not very suitable as substrates for use in test carriers since resorufin formed from them bleeds out, because of its water solubility.
Hydrolase substrates such as, for example methylumbelliferone galactoside, in which hydrolysis causes only a change of the fluoresence properties, are also unsuitable for a visual evaluation. Methylumbelliferone galactoside also gives rise to a substance, which easily bleeds out of the test strips.
Furthermore, processes for the detection of substances with hydrolase activity have been suggested in which uncolored hydrolase substrates are used which, in the case of reaction with a hydrolase, are converted into colored products. Consequently, in the case of these detection reactions, the formation of a color is to be assessed rather than a color change. This can take place in a simple way by comparison of the color intensity with a color shade of a color scale. Even color blind persons can evaluate such a test.
5-Bromo-4-chloro-3-indolyl-.beta.-galactoside is such a substrate. After cleavage and oxidation, this substrate gives a colored material which does not bleed out on test carriers. However, the substrate itself is not very water-soluble. Thus, a test constructed thereon is not very suitable for a rapid visual evaluation. The mechanism which leads to the color formation is the oxidative dimerisation of two 5-bromo-4-chloroindolyl units to give an indigo colored material. Thus, for the formation of one molecule of colored material, two enzymatic cleavages are necessary. Thus, only half of the theoretically possible sensitivity is achieved.
In Biochem. Z., 333, 209/1960, compounds were suggested as substrates in which the hydrolysis thereof liberates phenols. However, these easily bleed out of the test carrier. In the case of nitrophenol, during the indicator reaction, a more or less intensive yellow coloration develops. The visual evaluation in the range of small concentrations is very unfavorable at the wavelengths of the nitrophenols.
For improving visibility, in an additional reaction, the liberated phenols are coupled oxidatively with 4-aminoantipyrine or methylbenzothiazolinone hydrazone (Anal. Biochem., 40, 281/1971) or with diazonium salts to give azo dyes (see, for example, Histochemie, 37, 89/1973). Some of these dyes are admittedly suitable for use in test carriers since they do not bleed out very easily. However, the use of these coupling reactions is disadvantageous for other reasons. Thus, for carrying out the test on a fleece or film, all the reagents necessary for this additional reaction must also be applied to a fleece or introduced into a film. The large number of reagents, for example coupling partners, make the carrying out of the test complicated and result in numerous disadvantages and difficulties. Thus, in individual cases, care must be taken that the reagents do not already react prematurely with one another or non-specifically with other components of the sample solution etc. Thus, aminoantipyrine or methylbenzothiazolinone hydrazone can also react with components in, for example, investigated urine and thus falsify the measurement. Other coupling components, for example diazonium salts, considerably impair the storage stability. In choosing reagents, it should also be required that they not themselves have an inherent color, or other disadvantages.
Bleeding phenomena which occur in the case of some of the colored materials formed on test carriers will, for example, result in color intensity not being equal in all points of the fleece or film. This results in disadvantages in the evaluation. Since, for example, the measurement values are falsified.
Therefore, there is a need for hydrolase substrates in which the above-mentioned disadvantages are avoided and with the help of which substances with hydrolase activity can also be detected on test carriers in a simple, rapid and dependable manner.
It is an object of the present invention to satisfy this need.